Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone

K, Osaka; Takakura, Tadakazu; Narukawa, Kayo; Takahata, Masahiro; Endo, Kenji; Kiyota, Hiroshi; Onodera, Satoshi

doi:10.1007/s10156-008-0610-7

Cited by 78 publications

(66 citation statements)

References 30 publications

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“…The remaining four strains with a mosaic penA gene had raised CFX MICs of 0.064 to 0.094 g/ml, whereas all other nonmosaic isolates tested exhibited MICs of Յ0.047 g/ml. Furthermore, all 16 strains harboring a mosaic penA allele also showed raised CRO MICs in the range of 0.023 to 0.094 g/ml, which were noticeably higher than those of strains with nonmosaic patterns, in agreement with previously reported observations (37,40,41).…”

Section: Resultssupporting

confidence: 91%

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

et al. 2016

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Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10 3 to 10 4 genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. G onorrhea is the second most common bacterial sexually transmitted infection worldwide, with an estimated 78 million new cases in 2012 (1). Moreover, Neisseria gonorrhoeae has developed resistance to most current and past treatment options. Antimicrobial-resistant (AMR) gonorrhea is a major public health concern about which the World Health Organization (WHO) emphasizes the importance of global surveillance to identify emerging resistance, monitor trends, and inform revisions of treatment guidelines (2, 3).At a molecular level, the mechanisms that confer resistance to the most common treatment options have been well characterized. For instance, the acquisition of mosaic penA alleles, with or without substitutions at amino acid position 501 of the encoded penicillin-binding protein 2 (PBP2), has been linked to decreased susceptibility or resistance to the extended-spectrum cephalosporins (ESCs) cefixime (CFX) and ceftriaxone (CRO) (4, 5). In particular, strains harboring a mosaic pattern XXXIV penA gene, including the internationally spreading N. gonorrhoeae multiantigen sequence typing (NG-MAST) genogroup 1407, have been responsible for ESC treatment failures in severa...

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Section: Resultssupporting

confidence: 91%

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

et al. 2016

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“…Specifically, we detected an A501V mutation of penA, which contributes to decreased susceptibility to CRO (38,39). The A501V mutation that arose spontaneously in our study was also observed to arise spontaneously in a separate study conducted by Takahata et al (40).…”

Section: Discussionsupporting

confidence: 75%

Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

Gong

Lai

Liu

et al. 2016

Antimicrob Agents Chemother

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dThe emergence of ceftriaxone-resistant Neisseria gonorrhoeae is currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance in Neisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation in penA (A501V). However, differential analysis of the genomes and transcriptomes between pre-and postselection strains revealed many other mutated genes as well as up-and downregulated genes. Transformation of the mutated penA gene into nonresistant strains increased the MIC between 2.0-and 5.3-fold, and transformation of mutated ftsX increased the MIC between 3.3-and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, while pilM, pilN, and pilQ were downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance of Neisseria gonorrhoeae to ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. Gonorrhea is one of the most prevalent sexually transmitted infections (STIs), and it is associated with many severe clinical symptoms such as prostatitis, seminal vesiculitis, pelvic inflammation, infertility, and ophthalmia neonatorum (1). Although epidemiological data show a decreased trend in gonorrhea incidence as a whole, in some countries, the incidence is increasing, especially in specific population groups (adolescents, men who have sex with men, or sex workers) or specific venues (brothels) (2-5). The pathogen responsible for this disease is Neisseria gonorrhoeae. According to worldwide surveillance reports, N. gonorrhoeae has developed resistance to several kinds of antibiotics over the past decades (6-10). Of increasing concern is the prevalence of ceftriaxone (CRO)-resistant N. gonorrhoeae (11), because CRO is the first-line empirical drug for gonorrhea treatment in dual therapy with azithromycin or doxycycline recommended by the WHO (covering the Western Pacific region and the Southeast Asian region) and the CDC (12-14). Several CRO-resistant gonococcal strains, such as H041 and F89, were isolated recently from patients who had a treatment failure with CRO (H041) and cefixime (F89) (15-18). Highlighting the seriousness of these resistant strains, Ohnishi et al. proposed the concept of an N. gonorrhoeae "superbug" in 2011 (17). Uncovering the mechanisms of CRO resistance is now a critical topic of N. gonorrhoeae research.Previous s...

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“…The primary mechanism for ESC-DS is modification of the penA gene (penicillin binding protein 2 [PBP2]) including various mutations of the wild-type gene and a recombinant mosaic allele containing portions of genetic sequences from commensal Neisseria (3,4,9,(15)(16)(17)(18). Mutations in the promoter and/or the coding region of the repressor gene mtrR cause overexpression of the MtrCDE efflux pump (3,4,15,19,20) and porB1b gene mutations (penB G120 and/or A121 mutation) have been shown to cause decreased membrane permeability and contribute to decreased susceptibility toward cephalosporins (3,4,15,(19)(20)(21)(22).…”

mentioning

confidence: 99%

Whole-Genome Phylogenomic Heterogeneity of Neisseria gonorrhoeae Isolates with Decreased Cephalosporin Susceptibility Collected in Canada between 1989 and 2013

et al. 2015

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A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections. N eisseria gonorrhoeae is a Gram-negative diplococcus bacterium that causes gonorrhea infections. Gonorrhea is the second most reported bacterial sexually transmitted infection (STI) in Canada, with reported cases increasing from 15.5 per 100,000 in 1997 to 36.2 per 100,000 in 2012 (1), and approximately 106 million cases are estimated annually worldwide (2). N. gonorrhoeae bacteria have developed resistance against sulfonamides, penicillins, tetracyclines, and fluoroquinolones (3, 4), and current treatment options now include third-generation extended-spectrum cephalosporins (ESC), namely, cefixime (CFM) and ceftriaxone (CRO) (5). MIC creep has seen the modal MIC values rise between 2001 and 2010 in Canada from 0.016 g/ml to 0.125 g/ml and 0.063 g/ml for CFM and CRO, respectively (6). These results coincide with recent clinical reports of treatment failures to primarily CFM monotherapy in Canada (7,8) and additional global reports of high-level CRO MICs in isolates from Japan and Europe (9-13). Furthermore, isolates with decreased susceptibility to cephalosporins and coresistance to azithromycin (AZM), a recommended cotherapy (5), have recently been identified in Canada (14).Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) and resistance to AZM have been attributed to several molecular mechanisms. The primary mechanism for ESC-DS is modification of the ...

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Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone

Cited by 78 publications

References 30 publications

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

Whole-Genome Phylogenomic Heterogeneity of Neisseria gonorrhoeae Isolates with Decreased Cephalosporin Susceptibility Collected in Canada between 1989 and 2013

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